Allosteric transition intermediates modelled by crosslinked haemoglobins

Abstract

THE structural end-points of haemoglobin's transition from its low-oxygen-affinity (T) to high-oxygen-affinity (R) state, have been well established by X-ray crystallography^1á€-7, but short-lived intermediates have proved less amenable to X-ray studies. Here we use chemical crosslinking to fix these intermediates for structural characterization. We describe the X-ray structures of three haemoglobins, α₂β¹S⁸²β, α₂β¹Tm⁸²β and α₂β^1,82Tm⁸²β, which were crosslinked between the amino groups of residues βVal1 and βLys82 by 3,3′-stilbenedicarboxylic acid (S) or trimesic acid (Tm) while in the deoxy state, and saturated with carbon monoxide before crystallization. α₂β¹S⁸²β, which has almost normal oxygen affinity, is completely in the R-state conformation; however, α₂β¹Tm⁸²β and α₂β^1,82Tm⁸²β, both of which have low oxygen affinity, have been prevented from completing their transition into the R state and display many features of a transitional intermediate. These haemoglobins therefore represent a snapshot of the nascent R state.